Adenoviral vectors have held a prominent position in efforts to deliver therapeutic genes to cells, including muscle. However, a major limitation to first generation adenoviral vectors has been the aggressive immune response that these vectors elicit in vivo. We have recently described the design and construction of a novel adenoviral vector that appears to circumvent many of the immunological problems encountered by previous vectors. The 'helper-dependent' system expresses no virally-encoded antigens, and has a very large carrying capacity for DNA. High-level, muscle-specific murine dystrophin expression in mdx mouse muscle was demonstrated using one such recombinant vector. However, the longevity of dystrophin expression was limited by immune responses to beta-galactosidase that was co-expressed by the vector. The next generation of dystrophin-expressing, high capacity adenoviral vectors eliminates the lacZ gene cassette and uses novel helper virus technology to further decrease helper virus in the purified vector preparation. In this grant proposal, critical aspects of gene delivery, persistence and therapeutic effect will be studied in order to accomplish long-term genetic complementation of dystrophin-deficient muscle. Effects on muscle function will be assayed biochemically, histologically and physiologically. In Aim 1 the immunological effects of gene delivery on homologous proteins will be explored by cross- species dystrophin gene delivery. In Aim 2 novel strategies of adenoviral vector delivery, including muscle-specific targeting, will be tested. Aim 3 investigates the physiological benefits of full-length dystrophin delivery using a high-capacity adenoviral vector. Mouse age and immunosuppressive treatment are variables that will be tested for their effects on indices on muscle function afforded by adenoviral vector-mediated dystrophin delivery. In Aim 4, the biochemical, functional and immunological benefits of adenoviral vector-mediated gene transfer to fetal muscle will be investigated.